This disclosure is concerned generally with the immobilization of enzymes using porous, high surface area, water insoluble support materials. The disclosure is specifically concerned with a novel method of recycling the support material of spent immobilized enzyme composites so that the material can be conveniently re-used for enzyme immobilization.
It is known that enzymes can be immobilized by various means using a wide variety of water-insoluble support materials. See, for example, U.S. Pat. No. 3,645,852, to Axen et al. (chemical bonding of enzymes to organic support materials), U.S. Pat. No. 3,519,538 to Messing et al. (chemical bonding of enzymes to inorganic supports), U.S. Pat. No. 3,850,751, to R. A. Messing (adsorption of enzymes to inorganic supports), and U.S. Pat. No. 3,705,084 to Reynolds (chemical bonding to supports including both organic and inorganic materials). In general, many preferred support materials (carriers) are porous and/or have a relatively high surface area (e.g. &gt; 5 m.sup.2 /g) which permits a relatively large amount of enzyme to be loaded onto the carrier.
Even though numerous support materials have been successfully used to prepare immobilized enzyme composites having relatively high enzyme loadings and long enzymatic half-lives, it can be appreciated that the use of such composites, especially the continuous use, is economically time-limited. Regardless of the loading or enzymatic half-life of a given immobilized enzyme composite, it is known that, in use, the total enzymatic activity tends to decline with time. Thus, at a given point in time, it becomes more economical to simply replace "spent" enzyme composite with "fresh" composite.
As used herein, the expression "spent" immobilized enzyme composite, or its equivalent, refers to an immobilized enzyme composite which, after at least some use for its intended purpose, has become uneconomical to use further. The expression "fresh" immobilized enzyme composite refers to such composite which can still be economically used for its intended purpose. Several factors may determine the point in use or time when a given immobilized enzyme system becomes "spent" or uneconomical to use. For example, the enzymatic half life or amount of active enzyme on or within the support may have dropped to a relatively low level. The composite may have become contaminated with microbial growth which precludes further economical use. The composite may become contaminated with an undesirable excess of various metal ions which become associated with the composite after prolonged, continuous use with a substrate solution to which various buffers containing such ions are often added. The enzymes may simply become inactive. Regardless of cause or causes, it can be appreciated that a spent composite can contain a variety of materials both organic (e.g. proteins, microbes) and inorganic (e.g. salts). For purposes of this disclosure, all such materials associated with a spent composite are referred to as contaminants and they include substantially all materials except the support itself.
Although the support materials used for many immobilized enzyme systems are relatively inexpensive and may be discarded after use or when the immobilized enzyme composite is deemed spent, it can be appreciated that in some cases the reuse or recycling of the support materials may offer distinct advantages. For example, the reuse of support materials would not only offer possible cost savings, but also help avoid problems associated with the discharge of spent composites.
It is known that various pyrolysis treatments can be used to burn off organic materials on inorganic supports. However, as pointed out in U.S. Pat. No. 3,965,035, simple pyrolysis does not assure the removal of all contaminants (e.g., various metal ions from substrate solutions) which tend to minimize subsequent enzyme reloading and half life. In the above-cited patent, a two-step method of regenerating an inorganic enzyme carrier is disclosed. In the first step, the spent enzyme composite is pyrolyzed at a temperature ranging from about 500.degree. to 900.degree. C under conditions sufficient to assure removal of substantially all carbonaceous matter. Then, the carrier is reacted with a neutralized citrate solution to assure removal of remaining contaminants. Although the two-step method is quite effective in permitting the recycling of the specific carriers disclosed, it can be appreciated that the method is somewhat cumbersome in that the pyrolysis step generally requires removal of the spent composite from its container (e.g. a flow-through column) and placement in an appropriate furnace, followed by removal from the furnace, replacement in the column and treatment with the citrate solution.
In copending patent application Ser. No. 597,012 entitled "Enzyme Carrier Regeneration" we disclosed that the two-step process of U.S. Pat. No. 3,965,035, could be replaced by a relatively simple one-step regeneration technique which did not require a pyrolysis step. The one-step method involved reacting a specific MgO-Al.sub.2 O.sub.3 support for the enzyme glucose isomerase with a solution of sodium hypochlorite under conditions sufficient to assure removal of all contaminants. Since the disclosure of our copending application, we have found that the disclosed single step technique can be used to recycle other enzyme support materials and thereby provide a relatively simple, economical method of regenerating enzyme supports and preparing fresh immobilized enzyme composites. Details of our methods are disclosed herein.